AIDS (acquired immunodeficiency syndrome) is a deadly disease caused by the human immunodeficiency virus (HIV) which is a retrovirus. Despite intense research for nearly twenty years, a cure for AIDS has not yet been developed. Present treatments increase the life expectancy of AIDS patients, but extremely high mortality rates continue.
The expression of human immunodeficiency virus type 1 (HIV-1) is controlled by a post-transcriptional mechanism. From a single primary transcript several mRNAs are generated. These RNAs can be divided into three main classes: unspliced 9-kb, singly spliced 4-kb and the multiply spliced 2-kb RNAs. Each of these RNAs is exported to the cytoplasm for translation and, in the case of the 9 kb RNA, for packaging into virions (Kingsman and Kingsman, 1996; the publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References). Normally, pre-mRNAs must undergo a splicing process to remove one or more introns before being exported to the cytoplasm. HIV overcomes this limitation, allowing singly spliced and unspliced RNA to be exported via interaction with its own encoded Rev protein. Rev is responsible for the expression and cytoplasmic accumulation of the singly spliced and unspliced viral mRNAs by direct interaction with a target sequence (Rev response element or RRE) present in these mRNAs. This regulatory protein binds an RNA stem-loop structure (the RRE) located within the env coding region of singly spliced and unspliced HIV RNAs (Zapp and Green, 1989; Cochrane et al., 1990; D'Agostino et al., 1995; Malim et al., 1990). Binding of Rev to this element promotes the export, stability and translation of these HIV-1 RNAs (Arrigo and Chen, 1991; D'Agostino et al., 1992; Emerman et al., 1989; Feinberg et al., 1986; Felber et al., 1989; Hammarskjold et al., 1989; Lawrence et al., 1991; Schwartz et al., 1992; Malim et al., 1989; Favaro et al., 1999; Hope, 1999). The export process is mediated by the nuclear export signal (NES) of Rev which is a leucine rich region which binds the receptor exportin 1/CRM1 which mediates the export of the viral RNA. It is believed that CRM1 bridges the interaction of Rev with the nucleoporins required for export to the cytoplasm (Hope, 1999).
When Rev and Tat are expressed independently of other HIV transcripts, these proteins localize within the nucleolus of human cells (Cullen et al., 1988; Luznik et al., 1995; Dundr et al., 1995; Endo et al., 1989; Siomi et al., 1990; Stauber and Pavlakis, 1998). The simultaneous presence of a nuclear export signal (NES) as well as a nuclear import/localization signal (NLS) confers upon Rev the ability to shuttle between the nucleus and the cytoplasm (Hope, 1999). The Rev protein preferentially accumulates in the nucleolus in Rev-expressing cells (in the absence of RRE-containing RNA) and in the early phase of HIV infection (Dundr et al., 1995; Luznik et al., 1995). The reason for this specific sub-cellular localization is unknown. One possibility is that the nucleolus functions as the storage site for the Rev protein. Another and more compelling alternative, is that the Rev protein moves from the nucleus to the cytoplasm through the nucleolus. There is evidence that in the nucleolus Rev recruits nucleoporins Nup 98 and Nup 214 via hCRM1 (Zolotukhin and Felber, 1999). These results suggest a Rev-hCRM1-nucleoporins committed or pre-committed cytoplasmic export complex assembles in the nucleolus, and that the nucleolus can play a critical role in the Rev function.
To date, published data concerning nucleolar localization of HIV-1 RNAs are inconclusive. Using electron microscopy and in situ hybridization, Romanov et al. (1997) detected a subgenomic mRNA expressing the HIV-1 p37gag (containing the RRE element) in all the sub-cellular compartments (including the nucleoli) of HL Tat cells. Interestingly, they observed that the expression of Rev induced relocalization of HIV RNAs into two nonrandom patterns. One of these, the long track in the nucleoplasm, was radially organized around and in contact with the nucleoli. Other investigators using in situ hybridization analyses performed on mammalian cell lines transfected with different HIV-1 subgenomic or genomic constructs failed to detect HIV-1 RNA in the nucleolus (Zhang et al., 1996; Boe et al., 1998; Favaro et al., 1998; Favaro et al., 1999). The discrepancy in these results might be due to the different HIV-1 constructs, cell lines, and in situ hybridization protocols used by the various investigators. Furthermore, it should be taken into consideration that RNA export is a dynamic process; the rate of export as well as the amount of the HIV-1 RNA passing through the nucleolus can be limiting factors for in situ hybridization-mediated detection of nucleolar localized transcripts.
Ribozymes are RNA molecules that behave as enzymes, severing other RNAs at specific sites into smaller pieces. The hammerhead ribozyme is the simplest in terms of size and structure and can readily be engineered to perform intermolecular cleavage on targeted RNA molecules. These properties make this ribozyme a useful tool for inactivating gene expression, ribozymes being very effective inhibitors of gene expression when they are colocalized with their target RNAs (Sullenger and Cech, 1993; Samarsky et al., 1999). They may be valuable therapeutic tools for repairing cellular RNAs transcribed from mutated genes or for destroying unwanted viral RNA transcripts in the cell. However, targeting ribozymes to the cellular compartment containing their target RNAs has proved a challenge. Now, Samarsky et al. (1999) report that a family of small RNAs in the nucleolus (snoRNAs) can readily transport ribozymes into this sub-cellular organelle.
Small nucleolar RNAs (snoRNAs) are small, stable RNAs that stably accumulate in the nucleolus of eukaryotic cells. There are two major classes of snoRNA, each with its own highly conserved sequence motif. Both classes are involved in the post-transcriptional modification of the ribosomal RNA. The C/D box snoRNAs regulate 2′-O-methylation of the ribose sugars of ribosomal RNAs (rRNAs), and the H/ACA box snoRNAs guide pseudouridylation of rRNA uridine bases. A few snoRNAs also participate in processing precursor rRNA transcripts (Lafontaine and Tollervey, 1998; Weinstein and Steitz, 1999; Pederson, 1998). Most snoRNAs are transcribed and processed in the nucleus, although some may be synthesized in the nucleolus (the nuclear site of rRNA synthesis). It has been reported that the C and D boxes are important for stability, processing and nucleolar localization. In particular it has been demonstrated that an artificial RNA bearing the two boxes can be delivered into the nucleolus.
Samarsky et al. chose yeast for their experiments because the requirements for trafficking of a specific snoRNA (called U3) are well understood in this organism. They showed that nucleolar localization of the yeast U3 snoRNA was primarily dependent on the presence of the C/D box motif (Samarsky et al., 1998). The investigators appended a test ribozyme to the 5′ end of U3, and then inserted its RNA target sequence into the same location in a separate U3 construct. So both the ribozyme and its target were expressed in separate, modified U3 snoRNAs. The snoRNA-ribozyme molecule (called a snorbozyme) and its U3-tethered target were transported into the nucleolus. Here the ribozyme cleaved its target RNA with almost 100% efficiency.
Three crucial prerequisites for effective ribozyme action are (i) colocalization of the ribozyme and its RNA target in the same place, (ii) accessibility of the cleavage site in the target RNA to pairing with the ribozyme, and (iii) high levels of ribozyme relative to target RNA (Sullenger and Cech, 1993; Lee et al., 1999). The importance of colocalization was first demonstrated by tethering a ribozyme to the packaging signal (psi) of a murine retroviral vector and showing that copackaging of the ribozyme with a psi-tethered target resulted in greater than 90% reduction in viral infectivity (Sullenger and Cech, 1993).
Samarsky and colleagues used a clever method to assay ribozyme activity based on the rate of appearance of one of the two cleavage products (see the figure in Rossi, 1999a). The RNA target tethered to U3 is stable, with a half-life of over 90 minutes, and its cleavage by the ribozyme am generates two products: a short, rapidly degraded 5′ fragment and a 5′ extended form of the U3 snoRNA. The 5′ extension itself gets degraded, leaving intact the U3 hairpin, which is quite stable and easily distinguished from endogenous U3. Taking advantage of the accumulation of this stable product, the investigators were able to measure the kinetics of ribozyme cleavage in vivo. By using similar assay systems, it is possible to analyze ribozyme cleavage kinetics for virtually any ribozyme-substrate combination under physiological conditions.
There are plenty of applications for snorbozymes, particularly as the nucleolus is proving to be more than just the place where rRNA is synthesized. For example, precursor transfer RNAs (Bertrand et al., 1998), RNA encoding the enzyme telomerase, signal recognition particle RNAs, and U6 snRNAs all pass through the nucleolus where they are either processed or receive base and/or backbone modifications (Weinstein and Steitz, 1999). Several RNAs have been reported to pass through the nucleolus for processing, particle assembly, or other modification (Pederson, 1998). These include c-myc, N-myc, and myoD1 mRNAs (Bond and Wold, 1993), the signal recognition particle RNA (Jacobson and Pederson, 1998; Politz et al., 2000),U6 small nuclear RNA (Tycowski et al., 1998), some pre-tRNAs in yeast (Bertrand et al., 1998), and the RNAse P RNA (Jacobson et al., 1997). There is also evidence that telomerase RNA is processed within the nucleolus (Mitchell et al., 1999; Narayanan et al., 1999b). Transcription and replication of the neurotropic Borna disease virus have also been shown to occur within the nucleolus (Pyper et al., 1998). Importantly, the HTLV-1 env RNAs have been demonstrated to be partially localized in the nucleolus (Kalland et al., 1991). HTLV-1 and HIV-1 have a similar posttranscriptional regulation mechanism, and the Rex protein, a functional homolog of HIV-1 Rev, also has nucleolar localization properties. Viral proteins such as HIV's Rev and Tat and HTLV-1's Rex accumulate in this sub-cellular organelle (Stauber and Pavlakis, 1998; Siomi et al., 1988; Cullen et al., 1988). Rev is a crucial regulatory protein that shuttles unspliced viral RNA from the nucleolus into the cytoplasm. Recent findings show that Rev itself is transported out of the nucleolus by binding to a Rev-binding element in a U16 snoRNA (Buonomo et al., 1999). Using a snoRNA to localize a ribozyme that targets viral RNA to the nucleolus may be an effective therapeutic strategy to combat HIV. Ribozymes, antisense RNAs, and RNA decoys that bind Rev or Tat may be more effective in the nucleolus than in other regions of the nucleus or cytoplasm. SnoRNA chimeras harboring ribozymes or protein-binding elements should prove valuable not only therapeutically but also for elucidating why certain RNAs and proteins traffic through the nucleolus.